1. Field of the Invention
The present invention relates to a method for electrophoresis separation and detection of substances in living bodies, such as DNA, and an apparatus for the electrophoresis separation and detection. It relates to, in particular, a method for electrophoresis analysis in which a plurality of fluorophore-labeled samples are analyzed in one and the same lane, and an apparatus for the electrophoresis analysis.
2. Description of the Related Art
With the advance of Human Genome Program and DNA diagnosis, there is a growing demand for rapid determination of the base sequences of two or more kinds of DNA's and examination using various DNA probes. In the determination of the base sequences of DNA's and the measurement for DNA diagnosis, there has come to be used a technique comprising separating fluorophore-labeled DNA's (the same applies also to substances other than DNA, such as sugars and peptides) by gel electrophoresis and measuring the migrating DNA's in real time. For increasing the number of samples which can be measured at the same time, there are, for example, the following requirements: (1) the number of lanes are increased, (2) the electrophoretic migration rate is increased to increase the throughput, and (3) more kinds of labeling fluorophores (a larger number of colors, i.e. fluorescence emission wavelengths) are used.
For assuring a large number of lanes, capillary array electrophoresis apparatus comprising a large number of capillaries with an inner diameter of about 1 mm put side by side have been developed as a substitute for conventional slab gels (JP-A-05-072177, U.S. Pat. No. 5,277,780 and U.S. Pat. No. 5,366,608). Capillary array electrophoresis method is suitable for satisfying the above-mentioned requirements (1) and (2).
On the other hand, in a method comprising labeling two or more kinds of DNA's with many kinds of fluorophores, four kinds of fluorophores are widely used in relation to DNA sequencers. As an exciting light, there are used Ar ion laser (488 nm or 515 nm), YAG laser (532 nm), etc. As the fluorophores, there are often used, for example, FAM (emission wavelength: about 520 nm), JOE (emission wavelength: about 549 nm), TAMRA (emission wavelength: about 575 nm) and ROX (emission wavelength: about 600 nm) which are available from the ABD division of The Perkin-Elmer Corporation, and there are also used FITC (emission wavelength: about 520 nm), Sulforhodamine 101 (emission wavelength: about 615 nm), Cy5 (emission wavelength: about 670 nm), etc. As a means for spectroscopic detection of fluorescences emitted by these fluorophores, there are, for example, time-sharing measurement with a rotating filter, use of an image splitting or dividing prism (JP-A-02-269936 and U.S. Pat. No. 5,062,942), application of wavelength dispersion by means of a prism (JP-A-01-116441 and U.S. Pat. No. 4,832,815) or a grating.
For detecting fluorescences emitted by fluorophores while distinguishing them from one another, it is necessary that the maximum emission wavelengths of the fluorophores should be different from one another by about 30 nm or more. The wavelength region in which the fluorescences are observed is 500 nm to 700 nm, so that only 7 to 8 kinds of fluorophores having different emission wavelengths can be used. Moreover, the number of kinds of fluorophores efficiently excitable by exciting light having one wavelength is 2 or 3. When more kinds of fluorophores are used, two or more kinds of lasers having different wavelengths should be used for exciting the fluorophores. In this case, since the exciting light should be prevented from being received by a photodetector, fluorophores having a maximum emission wavelength near the wavelength of the exciting light are not usable. Therefore, the number of kinds of practically usable fluorophores is limited to about 6.
Kambara and Nagai among the present inventors made the following attempt: two or more kinds of exciting lights having different wavelengths are casted on different places of a gel electrophoresis plate at a definite distance from one another, and the thus obtained fluorescence images are projected on different positions of a photodetector, whereby a plurality of samples are measured at the same time (JP-A-03-293557 and U.S. Pat. No. 5,307,148). However, when too many kinds of fluorophores are used in this method, the detection of fluorescences emitted by all the fluorophores becomes difficult. Therefore, the number of kinds of fluorophores used in this method is limited to 5 or 6. There has recently been proposed a method in which fluorescence is efficiently excited by utilizing excitation energy transfer between two kinds of the fluorophores (JP-A-05-60698 and Anal. Biochem. 231, 131-140 (1995). In addition, as to the efficiency of the energy transfer between the two kinds of the fluorophores as an energy donor and an energy acceptor, respectively, the equation set up by Th. Foerster is known (Annalen der Physik, 6. Folge. Band. 2. (1948) pp. 55-75). According to this reference, the energy transfer rate is inversely proportional to the sixth power of the distance between the energy donor and the energy acceptor and is proportional to the degree of overlapping of an emission spectrum of the energy donor with an absorption spectrum of the energy acceptor. The energy transfer rate is affected also by orientation between the energy donor and the energy acceptor.
L. Stryer and P. H. Haugland have reported experimental investigation on the dependence of the energy transfer on the distance between the energy donor and the energy acceptor (Proc. Natl. Acad. Sci. USA 58 (1967) pp. 719-726). They have revealed good conformity of the distance dependence of the energy transfer with the equation of Foerster by varying the distance between the energy donor and the energy acceptor which are located at the ends of a polyp eptide oligomer, by varying the number of the peptides. They obtained energy transfer efficiency values of 100% and 16% at distances between the energy donor and the energy acceptor of 1.2 nm and 4.6 nm, respectively.